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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
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  • A61K38/00—Medicinal preparations containing peptides
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• • A—HUMAN NECESSITIES
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  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/06—Tripeptides
  • A61K38/063—Glutathione
• • A—HUMAN NECESSITIES
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  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P39/00—General protective or antinoxious agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P39/00—General protective or antinoxious agents
  • A61P39/06—Free radical scavengers or antioxidants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/30—Hormones
  • C12N2501/38—Hormones with nuclear receptors
  • C12N2501/39—Steroid hormones
  • C12N2501/392—Sexual steroids

Definitions

• a method for conferring cytoprotection on a population of cells that includes providing a polycyclic phenol compound, and an anti- oxidant compound; and administering the polycyclic phenol compound and the anti-oxidant compound to the population of cells at an effective dose so as to confer cytoprotection such that the combined cytoprotective effect of the polycyclic phenolic compound and the anti- oxidant compound is greater than the additive effect of each compound administered separately under otherwise similar conditions.
• a further embodiment of the invention is a method of conferring cytoprotection on a population of cells in a subject, including the steps of providing a combination therapeutic dose containing an anti-oxidant and a polycyclic phenolic compound in a pharmaceutical formulation; administering the formulation in an effective dose to the population of cells such that the cytoprotective effect of the combination in the subject is greater than the additive effect of each compound provided separately; and protecting cells in the subject that would otherwise deteriorate and die in the absence of the pharmaceutical formulation.
• the combined effect may be a 100-10,000 fold enhancement, more particularly a 100- 5,000 fold enhancement, more particularly a 1,000-5,000 fold enhancement of cytoprotection resulting from the combined effect of the polycyclic compound and the anti-oxidant compared with each compound separately.
• the polycyclic phenol compound is an estrogen compound more particularly an estrogen compound having insubstantial sex activity exemplified by the ⁇ isomer of estradiol.
• the anti- oxidant is selected from the group consisting of a thiol, a phenol, a spin trapping agent, an aromatic amine, a carotenoid, a flavonoid, a selenium, an aminosteroids and an ubiquinones.
• cytoprotection includes neuroprotection.
• Figure 1 shows the effect of glutathione (GSH) and 17 ⁇ -estradiol on neuronal cells (SK-N-SH cells) treated with neurotoxic ⁇ -amyloid protein ( ⁇ AP) where ⁇ AP is a peptide of 10 amino acids (amino acids 25-35), hereafter referred to as " ⁇ AP 25-35".
• GSH glutathione
• ⁇ AP neurotoxic ⁇ -amyloid protein
• 17 ⁇ -estradiol (2 nM) and GSH (3.25 ⁇ M) on the toxicity induced by ⁇ AP 25-35 (20 ⁇ M) on SK-N-SH cells (10 6 cells/ml) in different cell culture media lacking GSH in the cell culture recipe is shown.
• Cells were plated at 10 6 cells/ml and were exposed to vehicle, ⁇ AP 25-35, 17 ⁇ -estradiol, GSH, or a combination as indicated.
• Controls represent the mean value for the vehicle, GSH, 17 ⁇ -estradiol and GSH+17 ⁇ -estradiol groups; were pooled together after it was determined they were not statistically significant from each other. Depicted are the mean values ⁇ SEM for 4-5 wells/group. *p ⁇ 0.05 versus controls determined by ANOVA followed by Scheffe's post hoc test.
• Figure 2 shows the effect of GSH and 17 ⁇ -estradiol on live SK-N-SH cell number treated with neurotoxic ⁇ AP 25-35 in the presence or absence of a non-protective dose of 17 ⁇ -estradiol (2nM).
• Cells were plated at 10 6 cell/ml.
• the effect of varying concentrations of GSH in the presence of 17 ⁇ -estradiol were compared with cells treated with GSH in the absence of 17 ⁇ -estradiol (open triangles) . Depicted are the mean values ⁇ SEM for 6 wells per group.
• Figure 4 shows the effects of dose of 17 ⁇ -estradiol on the average number of rat primary cortical neurons per photographic field when subjected to a ⁇ AP 25-35 challenge in the presence and absence of a non-protective dose of GSH.
• Cells were plated and exposed to treatments as previously indicated at the doses noted. Depicted are the mean values ⁇ SEM for 4-7 wells per group.
• a comparison of the EC 50 values for the different dose response curves using the Mann- Whitney Rank Sum Test showed a p ⁇ 0.057.
• Figure 5 shows the effects of estratrienes in the presence and absence of GSH on the neurotoxicity induced by ⁇ AP(25-35) in HT22 cells.
• the symbols represent:
• Figure 6 shows the effects of 17 ⁇ -estradiol in the presence and absence of GSH on the neurotoxicity induced by ⁇ AP 1-40 in HT22 cells.
• D indicates the presence of GSH and ⁇ indicates the absence of GSH.
• the line represents the value of the control group (no ⁇ AP 1- 40).
• a preferred embodiment of the invention is directed to the novel finding that the cytoprotective effect of polycyclic phenol compounds is substantially enhanced when administered with at least one additional anti-oxidant compound.
• cytoprotection is defined here and in the claims as the protection of cells against cell death or cell damage that would otherwise occur in the absence of a protective agent, where the cell death or cell damage might be caused by any mechanical damage, nutritional deprivation including oxygen deprivation, trauma, disease processes, damage due to exposure to chemicals or temperature extremes, aging or other cause.
• estradien compound as used here and in the claims is defined as any of the structures described in the 11th Edition of "Steroids” from Steraloids Inc., Wilton N.H., here incorporated by reference. Included in this definition are isomers and enantiomers including non-steroidal estrogens formed by modification or substitution of the compounds in the
• estrogen compounds included in this definition are estrogen derivatives, estrogen metabolites and estrogen precursors as well as those molecules capable of binding cell associated estrogen receptor as well as other molecules where the result of binding specifically triggers a characterized estrogen effect. Also included are mixtures of more than one estrogen, where examples of such mixtures are provided in Table ⁇ below.
• 17 -E2, ⁇ -estradiol, 17 ⁇ -estradiol, ⁇ -17-E2 , 17 ⁇ -E2, E2, 17 ⁇ E2, ⁇ E2 as used here and in the claims are intended to be synonymous.
• ⁇ l7-E2, ⁇ - 17 -E2 , ⁇ -estradiol 17 ⁇ -estradiol , 17 ⁇ E2, ⁇ E2 as used here and in the claims are intended to be synonymous and correspond to the ⁇ isomer of 17 ⁇ -estradiol.
• E-3-ol as used here and in the claims represents estratriene-3-ol
• polycyclic phenolic compound, polycyclic compounds or polycyclic phenols as used here and in the claims are meant to be synonymous and are defined according to patent application Serial number 08/685,574 herein incorporated by reference and includes any compound having a phenolic A ring as defined below and may contain up to
• Cyclopentanophen (A) anthrene ring compounds having two, three or four rings and a hydroxyl group on carbons 1, 2, 3 and/or 4 of the A ring. Additionally, R groups selected from sodium, potassium and/or ammonium salts may be attached to the alpha or beta positions to replace hydrogen on any available carbon in the structure. Such compounds may be further substituted on the A ring so as to preserve a single-OH group and further form a pyridine, pyriazine, pyrimidine, s-triazine, v-triazne, or as-triazine.
• phenol A ring with possible additional substitutions listed above may further be linked to a six carbon ring having one or more of the following structures- benzene, cyclohexane,, 1, 2-pyran; 1, 4-Pyran, 1, 2-pyrone, 1, 4-pyrone; 1, 2-Dioxin; 1, 3-Dioxin
• Azole isopyrrole (isoazole); 3-isopyrrole (isoazole); Pyrazole (1, 2-Daizole); 2- isoimidazole (1, 3-isodiazole); 1, 2, 3-triazle; 1, 2, 4 triazole; 1, 2-diothiole; 1, 2, 3-oxathiole, isoxazole (furo(a) monozole); oxazole (furo(b) monazole); thiazole; isothiazole; 1, 2, 3- oxadiazole; 1, 2, 3, 4-oxatiazole; 1, 2, 3, 5-oxatriazole; 1, 2, 3-dioxazole; 1, 2, 4-dioxazole; 1, 3, 2-diaxazole; 1, 3, 4-dioxazole; 1, 2, 5-oxathiazole; 1, 3-oxathiole, cyclopentane.
• cyclopentanophen (a) anthrene ring compound is selected from the group consisting of 1, 3,5 (10), 6, 8-estrapentaen; 1, 3, 5 (10), 6, 8, 11-estrapentaen; 1, 3, 5 (10), 6,-estratetraen 1, 3, 5 (10), 7-estratetraen ; 1, 3, 5 (10) 8- estratetraen; 1, 3, 5 (10) 16-estratetraen; 1, 3, 5 (10) 15-estratetraen; 1, 3, 5 (10)- estratrien; 1, 3, 5 (10) 15- estratrien.
• anti-oxidant refers to any molecule that prevents oxidation of a particular substrate by a second molecule.
• examples of anti-oxidants included in the invention are the following:
• Thiols including glutathione, taurine, cystein, homocysteine, and ⁇ -lipoic acid; phenols: including probucol, salicylates, Trolox C, 3 ,4-dihydroxytoluene, 3 , 4- dihydroxycinnamic acid, nordihydroxyquaiarectic acid, 2", 4'-dihydroxyacetophenone, 2', 5'- dihydroxyacetophenone, 3' 4'-dihydroxyacetophenone, propylgallate; spin trapping agents including dimethyl- 1-pyrroline-N-oxide, N-tert-butyl- ⁇ -phenylnitrone; aromatic amines including promethazine, chlorpromazine, ethoxquin, allopurinol, uric acid; carotenoids including ⁇ -carotene, ⁇ -carotene, ⁇ -carotene, lypcopene, Caratol; flavonoids including (+)- catechin, di
• Polycyclic compounds having a terminal phenolic A ring in combination with anti-oxidants for enhancing cytoprotective potency in a svnergistic manner Polycyclic compounds having a terminal phenolic A ring in combination with anti-oxidants for enhancing cytoprotective potency in a svnergistic manner.
• an anti-oxidant in the extracellular milieu provides enhanced cytoprotection when administered to a subject together with a polycyclic compound having a phenolic A ring, such as estrogen, at physiologically and pharmacologically relevant doses.
• a polycyclic compound having a phenolic A ring such as estrogen
• the enhanced effect of the combination of polycyclic phenolic compounds and anti-oxidants, more particularly compounds having insubstantial sex activity in combination with anti-oxidant thiols, more particularly estrogen compounds having insubstantial sex activity and glutathione occurs to promote cell protection beyond that observed with any single compound.
• the combination of anti-oxidant and polycyclic phenolic compounds including estrogens has utility in protecting cells from damage resulting from any of a number of events that are known to be detrimental to the cell including chemical damage such as caused by free radicals, excitatory aminoacids and amyloid plaque.
• the above combination is more effective for neuroprotection than either anti- oxidant or polycyclic phenolic compound alone and therefore presents a novel therapeutic approach to treating neuron loss in human subjects that occurs in neurodegenerative diseases such as Alzheimer's disease.
• the combination therapy disclosed here has utility in treating other disease conditions which result from enhanced cell death including ischemia, trauma and aging.
• FIG. 5 shows increases in neuroprotective effect of 100- 600 fold for three different estrogen compounds.
• estratrienes exhibit approximately 1000 - 5000 fold enhancement in their cytoprotective effect when administered with the anti-oxidant, glutathione.
• the enhancement observed with different combinations of agents falls within the range of 100 fold to 10,000 fold.
• the synergistic effect of anti-oxidant and estrogen compound was further observed in cells that lacked an estrogen receptor.
• Figure 6 shows the synergistic effect of glutathione with 17 ⁇ -estradiol in HT-22 cells.
• the enhanced potency of estrogens may result from its ability to donate from several positions on the A ring (Jellnick and Bradlow, 1990).
• a relatively stable oxidized form of estradiol may result from this hydrogen ion donation.
• a glutathione peroxidase can regenerate the reduced form of estrogen.
• This enzyme relies on GSH as its substrate for donation of the hydrogen group back to estrogen, and thereby result in a synergy between the activity of the two molecules.
• estrogens interaction for this glutathione system is supported by two lines of evidence. First, there are no apparent interactions noted between estrogen and the other thiols tested, lipoic acid or taurine, or any other anti-oxidants, including ascorbic acid or atocopherol. It should be noted that while ⁇ -tocopherol is a powerful anti-oxidant in its own right, estrogen has been argued to be even more powerful. Second, the ability of oxidized glutathione to work in this system, argues that estrogen may be interacting with the glutathione peroxidase/reductase process.
• estrogen in cell membranes is a reflection of the amounts of estrogen introduced into the blood stream. If estrogen is available, it may insert into the cell membranes of any available cell without specificity thereby providing a cell protective effect. However, as the amounts of estrogen become limiting, estrogens may predominantly located in the membranes of cells bearing estrogen receptors.
• ⁇ - estradiol is a sex hormone and known to act through the estrogen receptor
• ⁇ -estradiol or estratriene-3-ol are generally believed to be biologically inactive according to the art.
• These compounds are all examples of polycyclic phenolic compounds.
• the invention encompasses the use of any of the class of polycyclic phenolic compounds independent of their ability to bind the estrogen receptor.
• estradiol conjugated to bovine serum albumin BSA
• BSA conjugation 17 ⁇ - estradiol-6-(carboxymethyl) oxime: BSA containing 20 ⁇ M 17 ⁇ -estradiol
• BSA containing 20 ⁇ M 17 ⁇ -estradiol BSA containing 20 ⁇ M 17 ⁇ -estradiol
• a pharmaceutical preparation including a polycyclic phenolic compound and an anti- oxidant may be used for treating subjects suffering from trauma, chronic degenerative diseases or acute disease such as induced by an ischemic attack. Examples include Alzheimer's disease, Parkinson's disease, stroke, ischemia, heart attack or angioplasty, or brain or spinal cord trauma, hypoglycemia, anoxia, burns or surgeries that result in the loss of nutrient flow to the tissues.
• Embodiments of the invention may further be applied to the procedure of tissue transplantation, prior, during or after removal or reperfusion of cells, tissues or organs or during storage of the cells, tissues or organs and is applicable to any of the cells in the body.
• the following examples are presented are not intended to be limiting but nonetheless effectively illustrate the novel biological effect of the invention.
• Example 1 The Synergistic Effect of an Anti-oxidant with a Polycyclic Phenolic Compound Using SK-N-SH Human Neuronal Cells
• Glutathione was selected as an anti-oxidant and 17 ⁇ -estradiol was selected as the polycyclic compound.
• the assay utilized the human neuroblastoma cells SK-N-SH. The cells were treated with toxins, known to cause cell death, found in amyloid plaque - ⁇ -AP(l- 40) and ⁇ -AP (25-35). The numbers following "AP" correspond to the size and type of protein fragment identified by amino acid residue number.
• Lyophilized ⁇ AP 25-35 (1 mg) (Bachem, Torrance, CA) was initially dissolved in 200 ⁇ l ddH20 and with the addition of 800 ⁇ l of PBS, rapid aggregation was observed.
• 17 ⁇ -estradiol (17 ⁇ -estradiol, Steraloids, Wilton, NH) was initially dissolved at 10 mg/ml in absolute ethanol (Fisher Scientific Inc., Orlando, FL) and diluted in cell culture media to obtain the necessary concentrations
• ⁇ -tocopherol acetate was initially dissolved in 200 ⁇ l absolute ethanol and diluted in cell culture media to the appropriate concentrations.
• Lipoic acid thiotic acid
• taurine (2-aminoethanoic acid)
• ascorbic acid ascorbic acid
• SK-N-SH Neuroblastoma Cell Culture SK-N-SH neuroblastoma cells were obtained from American Type Tissue Collection (Rockville, MD). Cell cultures were grown to confluency in RPMI-1640 media (Fisher Scientific, Inc.) supplemented with 10% charcoal/dextran-treated fetal bovine serum (FBS), (Hyclone®), Logan, UT), 100 U/ml penicillin G and 100 mg/ml streptomycin (Sigma Chemical Corp.) in monolayers in Corning 150 cm 2 flasks (Fisher Scientific, Inc.) at 37° C under 5 % CO 2 , 95 % air. Media was changed three times weekly. Cells were observed with a phase contrast microscope (Nikon
• SK-N-SH cells used in the following experiments were in passes 4 to 12. The growth media was initially decanted and the cells were rinsed with 0.02% EDTA for 30 min at 37 C. Cells were then counted on a Neubauer hemacytometer (Fisher Scientific) and resuspended in appropriate media. Studies were initiated by plating 1 x 10 6 cells per well in 24 well plates, allowing attachment in regular media and then decanting that media and replacing with the appropriate treatment after 4 h.
• ⁇ AP 25-35 (20 ⁇ M), 17 ⁇ -estradiol (0.002 - 200 nM ), GSH (0.0325-325 ⁇ M ), ⁇ -tocopherol acetate (50 ⁇ M), ascorbic acid (100 ⁇ M), lipoic acid (10 ⁇ M), taurine (5 mM), or a combination as indicated.
• the 20 ⁇ M concentration of ⁇ AP was selected as we have shown that it is near the LD 50 for this peptide (Green et al, 1996). Selection of anti-oxidant concentrations were made on the basis of preliminary dose-response evaluations used to identify the concentration at which neuroprotection was not obtained.
• SK-N-SH cell viability was determined utilizing the trypan blue exclusion method (Black and Berenbaum, 1964; Tennant, 1964). After 72 h of incubation, treatment media was decanted and cells were lifted by incubating with 0.2 ml 0.02% EDTA for 30 min at 37 °C. Cells were suspended by repeated pipetting. One-hundred ⁇ l aliquots from each cell suspension were incubated with 100 ⁇ l aliquots of 0.4 % trypan blue stain for 5 min at room temperature. All suspensions were counted on a Neubauer hemacytometer within 10 min of addition of trypan blue stain. Two independent counts of live and dead cells were made for each aliquot.
• SK-N-SH cells were treated with A ⁇ 25- 35(20 ⁇ M) a single concentration of glutathione (3.25 ⁇ M.) and a variable concentrations of 17 ⁇ -estradiol in the range of 0-200nM. After 72 hours incubation, the cells were stained with
• ⁇ -17 -E2 and E-3-ol estrogens both behaved similarly to with an ED 50 of 6 nM and 14 nM, respectively, in the presence of GSH and ED 50 of 1014 nM and 3683 nM, respectively, in the absence of GSH.
• Example 2 The synergistic neuroprotective effect of an anti-oxidant with a polycyclic Phenolic compound on murine neuronal cells that lack functional estrogen receptors (HT-22) using an in vitro assay.
• Example 3 The neuroprotective effect of estrogen compounds with an anti-oxidant on primary rat cortical neuron cultures.
• Treatments were made directly to primary cultures on culture day 10, maintaining a constant volume added regardless of treatment as follows: Controls: absolute ethanol and PBS, samples: ⁇ AP 25-35 (10 ⁇ M), 17 ⁇ -estradiol (0.02 nM-2 ⁇ M), GSH (3.25 ⁇ M), or combinations as indicated. Once treatments were added, primary cultures were incubated for an additional 24 h and viability determined using the LIVE/DEAD® viability/cytotoxicity kit (Molecular Probes, Eugene, OR) according to manufacturer's instructions. Live cells are distinguished by the presence of intracellular esterase activity which cleaves the calcein AM dye, producing a bright green fluorescence when excited.
• Ethidium homodimer enters cells with damaged membranes, and upon binding to nucleic acids, produces a red fluorescence. Both dyes are excited at 485 nm, and cultures plates were viewed with a fluorescent microscope (Nikon Diaphot-300). Three random fields were photographed, and the average number of live cells per field was determined by counting the number of bright green cells. Again, the ability of estradiol was evaluated in the presence and absence of GSH (3.25 ⁇ m) (Fig x). The addition of ⁇ AP 25-35 (lO ⁇ M) to primary cortical neurons resulted in a 39% to 40% reduction in the average number of viable cells per field in the absence and presence of GSH, respectively (Fig 4). When increasing doses of 17 ⁇ -estradiol were evaluated with ⁇ AP

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